Promoter methylation analysis of WNT/β-catenin signaling pathway regulators to detect adenocarcinoma or its precursor lesion of the cervix

Objective: Cervical adenocarcinoma (AdCA) and adenocarcinoma in situ (ACIS) are frequently missed in cytology-based screening programs. Testing for high-risk human papillomavirus (hrHPV) improves their detection, but novel ACIS/AdCA specific biomarkers are needed to increase specificity for these lesions. Novel markers may be deduced from the WNT/β-catenin signaling pathway, which is aberrantly activated during cervical carcinogenesis. Methods: Promoter methylation of nine WNT-antagonists (APC, AXIN2, DKK3, SFRP2, SFRP4, SFRP5, SOX17, WIF1 and WNT5A) was evaluated by methylation-specific PCR (MSP) on a small series of cervical tissue specimens, including AdCA and SCC. To estimate the diagnostic potential of the genes most frequently methylated in AdCA an extended series of ACIS, AdCA, CIN3, SCC, and normal cervical tissue specimens (n = 131) as well as 49 hrHPV-positive scrapings were analyzed by quantitative MSP (qMSP). Results: The frequency of DKK3 and SFRP2 methylation was significantly higher in AdCA compared to SCC, i.e. 82% vs. 18% (p < 0.01) and 84% vs. 39% (p < 0.01), respectively, while SOX17 methylation frequency was significantly higher in SCC than AdCA, i.e. 89% vs. 62% (p < 0.05). Methylation of WIF1 was common in both AdCA (71%) and SCC (54%). Methylation frequencies ranged from 4% to 55% in precursor lesions and from 0% to 5% in normal biopsies. When tested on HPV-positive cervical scrapings, qMSP of the best ACIS/AdCA discriminator genes, i.e. DKK3 and SFRP2, detected all women with underlying ACIS/AdCA, compared to 3% of controls. Conclusions: DKK3 and SFRP2 promoter methylation is highly indicative for the presence of ACIS/AdCA, thereby providing promising triage markers for HPV-positive women at risk of ACIS/AdCA.